Genetically altered alfalfa producing clovamide and/or related hydroxycinnamoyl amides

ABSTRACT

Two novel cDNAs for two different genes, HDT1 and HDT2, are isolated from red clover and sequenced. Both HDT1 and HDT2 encode hydroxycinnamoyl-CoA:L-DOPA/tyrosine hydroxycinnamoyl transferase (HDT) which enzymatically produces clovamide and/or related hydroxycinnamoyl amides. Clovamide and related hydroxycinnamoyl amides reduce post-harvest protein degradation. Genetically altered alfalfa plants containing an expression cassette containing a cDNA encoding HDT1 or HDT2 are generated. These genetically altered alfalfa plants produce hydroxycinnamoyl-CoA:L-DOPA/tyrosine hydroxycinnamoyl transferase, which in turn produces clovamide and/or related hydroxycinnamoyl amides.

BACKGROUND OF THE INVENTION

Field of the Invention

This invention relates genetically altered alfalfa plants that containheterologous cDNA encoding hydroxycinnamoyl-CoA:L-DOPA/tyrosinehydroxycinnamoyl transferase (HDT) and produce clovamide and/or relatedhydroxycinnamoyl amides. This invention also relates to the cDNAs thatencode HDT1 and HDT2 obtained from red clover, expression vectorscontaining the cDNAs, and the use of the cDNAs and/or expression vectorsto genetically modify alfalfa so that the modified alfalfa can produceclovamide and/or related hydroxycinnamoyl amides. This invention alsorelates to genetically altered alfalfa that have a phenotype involvingpost-harvest protein protection.

Description of Related Art

Clovamide (an amide formed between caffeic acid and L-DOPA[L-3,4-dihydroxyphenylalanine]) is one of two major o-diphenol compoundspresent in leaves of red clover. The other major o-diphenol compoundpresent in red clover leaves is phaselic acid (an ester formed betweencaffeic acid and malic acid). When oxidized by the endogenous polyphenoloxidase system (PPO), caffeic acid derivatives such as phaselic acid andclovamide constitute a natural system of post-harvest protein protectionfor forage crops. See U.S. Pat. No. 8,338,339.

Unfortunately, many important forages, like alfalfa, do not make PPO orthe o-diphenol compounds needed for this process. For alfalfa alone,post-harvest proteolytic losses upon harvest and storage as silage costU.S. farmers an estimated $100 million annually. Poor utilization ofdegraded forage protein by ruminant animals also results in release ofthe excess nitrogen into the environment as urea. Forages rich in PPOand o-diphenols appear to have reduced protein and lipid degradation inthe rumen, with the potential for additional nitrogen utilizationefficiency and improved lipid profiles of animal products, respectively(see, Lee et al., 2004, J. Sci. Food Agric. 84:1639).

Mature red clover leaves accumulate relatively high levels of twocaffeic acid derivatives: phaselic acid (an ester formed between caffeicacid and malic acid) (5 to 8 mmol/kg fresh weight [FW]), and clovamide(an amide formed between caffeic acid and the amino acid L-DOPA) (3 to 6mmol/kg FW) (Sullivan and Zeller, 2013, J. Sci. Food. Agri.93(2):219-26). Previously, a red clover gene (HCT2, Genbank EU861219)encoding a hydroxycinnamoyl-CoA:malate hydroxycinnamoyl transferase(HMT) was shown to be crucial for phaselic acid accumulation in redclover leaves (Sullivan and Zarnowski, 2011, Plant Physiol.155(3):1060-7).

Although in red clover, phaselic acid is a major hydroxycinnamoyl-malateester, expression of red clover HCT2 in alfalfa results in mostlyaccumulation of p-coumaroyl-malate and feruloyl-malate, compounds thatdo not function with PPO to preserve forage protein. See, Sullivan, M.,(2015) “Engineering alfalfa to accumulate useful caffeic acidderivatives and characterization of hydroxycinnamoyl-CoA transferasesfrom legumes” in The Phytochemical Society of North America, Aug. 8-12,2015(conferences.illinois.edu/psna/documents/PSNA_2015_Full_Program.pdf).This accumulation pattern in alfalfa is actually consistent with thein-vitro enzymatic properties of HCT2 gene product (HMT) wherebyp-coumaroyl-CoA and feruloyl-CoA donor substrates are preferred overcaffeoyl-CoA by five- to tenfold (see Sullivan and Zarnowski, 2011).

Thus, a need exists for genetically altered alfalfa plants that canproduce clovamide and/or related hydroxycinnamoyl amides which canprotect proteins from post-harvest degradation. Such a geneticallyaltered alfalfa plant must contain cDNA encoding the appropriate enzymewhich produces clovamide. Based on the research presented below, thatenzyme is termed hydroxycinnamoyl-CoA:L-DOPA/tyrosine hydroxycinnamoyltransferase (HDT).

BRIEF SUMMARY OF THE INVENTION

It is an object of this invention to have two novel and isolated cDNAs,each encoding an enzyme having hydroxycinnamoyl-CoA:L-DOPA/tyrosinehydroxycinnamoyl transferase (HDT) activity. It is another object ofthis invention that the cDNA for HDT1 has the sequence of SEQ ID NO: 1or a sequence that is at least 95% identical to SEQ ID NO: 1. It isanother object of this invention that the cDNA for HDT2 has the sequenceof SEQ ID NO: 3 or a sequence that is at least 95% identical to SEQ IDNO: 3. It is a further object of this invention to have expressioncassettes containing a promoter operably linked to one of these cDNAs.The promoter can be constitutive, inducible, or tissue specific.

It is a further object of this invention to have two novel proteins,hydroxycinnamoyl-CoA:L-DOPA/tyrosine hydroxycinnamoyl transferase,referred to as HDT1 and HDT2. The amino acid sequence of HDT1 is SEQ IDNO: 2 or a sequence that is at least 95% identical to SEQ ID NO: 2 (solong as the protein possesses hydroxycinnamoyl-CoA:L-DOPA/tyrosinehydroxycinnamoyl transferase activity). The amino acid sequence of HDT2is SEQ ID NO: 4 or a sequence that is at least 95% identical to SEQ IDNO: 4 (so long as the protein possesseshydroxycinnamoyl-CoA:L-DOPA/tyrosine hydroxycinnamoyl transferaseactivity). It is a further object of this invention to have cDNAs thatencode HDT1 and HDT2 proteins (or a protein that is at least 95%identical to SEQ ID NO: 2 or SEQ ID NO: 4 and which possesshydroxycinnamoyl-CoA:L-DOPA/tyrosine hydroxycinnamoyl transferaseactivity). It is another object of this invention to have expressioncassettes containing a promoter operably linked to one of these cDNAsthat encode HDT1 or HDT2 (or a polynucleotide that encodes a proteinthat is at least 95% identical to HDT1 or HDT2 and possesseshydroxycinnamoyl-CoA:L-DOPA/tyrosine hydroxycinnamoyl transferaseactivity). The promoter can be constitutive, inducible, or tissuespecific.

It is another object of this invention to have a genetically alteredalfalfa plant that has hydroxycinnamoyl-CoA:L-DOPA/tyrosinehydroxycinnamoyl transferase activity, the genetically alfalfa plantcontains a promoter operably linked to a heterologous cDNA (anexpression cassette) such that the heterologous cDNA encodes ahydroxycinnamoyl-CoA:L-DOPA/tyrosine hydroxycinnamoyl transferase. It isan object of this invention that the encoded protein can be HDT1 (SEQ IDNO: 2), HDT2 (SEQ ID NO: 4), or a protein having an amino acid sequencethat is at least 95% to SEQ ID NO: 2 or SEQ ID NO: 4. It is a furtherobject of this invention to have a pollen, seed, or cell from thisgenetically altered alfalfa plant. It also an object of this inventionto have a tissue culture of cells from this genetically altered alfalfaplant.

It is an object of this invention to have a genetically altered alfalfaplant that has hydroxycinnamoyl-CoA:L-DOPA/tyrosine hydroxycinnamoyltransferase activity, the genetically alfalfa plant contains a promoteroperably linked to a heterologous cDNA (an expression cassette). It isanother object of this invention that the cDNA has a sequence that canbe SEQ ID NO: 1, SEQ ID NO: 3, or a sequence that is at least 95%identical to SEQ ID NO: 1 or SEQ ID NO: 3 so long as the encoded proteincontains hydroxycinnamoyl-CoA:L-DOPA/tyrosine hydroxycinnamoyltransferase activity. It is a further object of this invention to have apollen, seed, or cell from this genetically altered alfalfa plant. Italso an object of this invention to have a tissue culture of cells fromthis genetically altered alfalfa plant.

It is an object of this invention to have a method of reducingpost-harvest protein degradation in a genetically alfalfa plant by (a)introducing a promoter operably linked to a heterologous cDNA into analfalfa plant to provide a genetically altered alfalfa plant, such thatthe heterologous cDNA encodes a hydroxycinnamoyl-CoA:L-DOPA/tyrosinehydroxycinnamoyl transferase which can have an amino acid sequence ofSEQ ID NO: 2, SEQ ID NO: 4, a sequence that is at least 95% identical toSEQ ID NO: 2, or a sequence that is at least 95% identical to SEQ ID NO:4, and (b) selecting the genetically altered alfalfa plant that containsthe heterologous cDNA and/or produces protein encoded therein, such thatthe protein encoded by the heterologous cDNA produced clovamide, atleast one related hydroxycinnamoyl amide, or a combination thereof. Theclovamide, at least one related hydroxycinnamoyl amide, or combinationthereof reduces post-harvest protein degradation in the geneticallyaltered alfalfa. It is a further object of this invention that the stepof introducing the heterologous cDNA into the alfalfa plant occurs viaintrogression, breeding, or transfecting an expression cassettecontaining the heterologous nucleotide and promoter into the alfalfaplant. It is another object of this invention the step of selecting thegenetically altered alfalfa plant occurs via marker assisted selection.It is another object of this invention that marker assisted selectioninvolves using primers having a sequence of SEQ ID NO: 9 and/or SEQ IDNO: 10 in a PCR reaction.

It is another object of this invention to have a method of reducingpost-harvest protein degradation in a genetically altered alfalfa plantby (a) introducing a promoter operably linked to a heterologouspolynucleotide into an alfalfa plant to provide a genetically alteredalfalfa plant, such that the heterologous polynucleotide has thesequence of SEQ ID NO: 1, SEQ ID NO: 3, a sequence that is at least 95%identical to SEQ ID NO: 1, or a sequence that is at least 95% identicalto SEQ ID NO: 3, and such that the heterologous polynucleotide encodes aprotein having hydroxycinnamoyl-CoA:L-DOPA/tyrosine hydroxycinnamoyltransferase activity, and (b) selecting the genetically altered alfalfaplant that contains the heterologous polynucleotide and/or produces theprotein having hydroxycinnamoyl-CoA:L-DOPA/tyrosine hydroxycinnamoyltransferase activity, such that the protein havinghydroxycinnamoyl-CoA:L-DOPA/tyrosine hydroxycinnamoyl transferaseactivity produces clovamide, at least one related hydroxycinnamoylamide, or a combination thereof. The clovamide, at least one relatedhydroxycinnamoyl amide, or combination thereof reduces post-harvestprotein degradation in the genetically altered alfalfa. It is also anobject of the invention that the introducing of the heterologouspolynucleotide occurs via introgression, breeding, or transfecting anexpression cassette containing the heterologous nucleotide and promoterinto the alfalfa plant. It is a further object of the invention that theselecting of the genetically altered alfalfa plant occurs via markerassisted selection. It is another object of this invention that markerassisted selection involves using primers having a sequence of SEQ IDNO: 9 and/or SEQ ID NO: 10 in a PCR reaction.

It is yet another object of this invention to have a method ofconstructing a genetically altered alfalfa plant that producesclovamide, at least one related hydroxycinnamoyl amide, or a combinationthereof by (a) introducing a promoter operably linked to a heterologouscDNA into an alfalfa plant to provide a genetically altered alfalfaplant, such that the heterologous cDNA encodes ahydroxycinnamoyl-CoA:L-DOPA/tyrosine hydroxycinnamoyl transferase havingthe amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 4, a sequence thatis at least 95% identical to SEQ ID NO: 2, or a sequence that is atleast 95% identical to SEQ ID NO: 4, and (b) selecting the geneticallyaltered alfalfa plant that contains the heterologous cDNA and/orproduces the protein encoded therein. Thehydroxycinnamoyl-CoA:L-DOPA/tyrosine hydroxycinnamoyl transferaseproduces clovamide, at least one related hydroxycinnamoyl amide, or acombination thereof. It is a further object of this invention that theintroducing the heterologous cDNA occurs via introgression, breeding, ortransfecting an expression cassette containing the heterologousnucleotide and promoter into the alfalfa plant. It is yet another objectof this invention that the selecting of the genetically altered alfalfaplant occurs via marker assisted selection. It is another object of thisinvention that marker assisted selection involves using primers having asequence of SEQ ID NO: 9 and/or SEQ ID NO: 10 in a PCR reaction.

It is yet a further object of this invention to have a method ofconstructing a genetically altered alfalfa plant that producesclovamide, at least one related hydroxycinnamoyl amide, or a combinationthereof by (a) introducing a promoter operably linked to a heterologouspolynucleotide into an alfalfa plant to provide the genetically alteredalfalfa plant, such that the heterologous polynucleotide has thesequence of SEQ ID NO: 1, SEQ ID NO: 3, a sequence that is at least 95%identical to SEQ ID NO: 1, or a sequence that is at least 95% identicalto SEQ ID NO: 3, and such that the heterologous polynucleotide encodes aprotein having hydroxycinnamoyl-CoA:L-DOPA/tyrosine hydroxycinnamoyltransferase activity, and (b) selecting the genetically altered alfalfaplant that contains the heterologous polynucleotide and/or produces theprotein encoded thereby and the protein havinghydroxycinnamoyl-CoA:L-DOPA/tyrosine hydroxycinnamoyl transferaseproduces clovamide, at least one related hydroxycinnamoyl amide, or acombination thereof. It is an object of this invention that theintroducing the heterologous polynucleotide occurs via introgression,breeding, or transfecting an expression cassette containing the promoterand heterologous nucleotide into the alfalfa plant. It is yet anotherobject of this invention that the selecting of the genetically alteredalfalfa plant occurs via marker assisted selection. It is another objectof this invention that marker assisted selection involves using primershaving a sequence of SEQ ID NO: 9 and/or SEQ ID NO: 10 in a PCRreaction.

It is another object of this invention to have a kit for determining ifan alfalfa plant contains cDNA for HDT1 or HDT2 and thereby produces ahydroxycinnamoyl-CoA:L-DOPA/tyrosine hydroxycinnamoyl transferase. Thiskit contains at least one pair of polynucleotides; an identifying dye;and instructions for using the at least one pair of polynucleotides,such that the pair of polynucleotides have the sequence of SEQ ID NO: 9and SEQ ID NO: 10, respectively; and such that if the alfalfa plantpossesses the polynucleotide sequence of SEQ ID NO: 9 or SEQ ID NO: 10,then the alfalfa plant contains either the HDT1 or HDT2 gene.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1A and FIG. 1B show the sequence alignment of HDT1 (SEQ ID NO: 1)and HDT2 (SEQ ID NO: 3). ATG and TGA codons are bolded and underlined.The non-identical nucleotides are highlighted in black.

FIG. 2 is sequence alignment of HDT1 (SEQ ID NO: 2) and HDT2 (SEQ ID NO:4) with the non-identical amino acids highlighted in black.

FIG. 3 shows the enzymatic reaction of a hydroxycinnamoyl-Coenzyme Acompound and a phenolic amino acid compound byhydroxycinnamoyl-CoA:L-DOPA/tyrosine hydroxycinnamoyl transferase (HDT)to produce clovamide (hydroxycinnamoyl-L-DOPA) and/or relatedhydroxycinnamoyl compounds.

FIG. 4A demonstrates that E. coli BL21/pET28-HDT1 possesseshydroxycinnamoyl-CoA transferase activity via the reverse phase HPLC ofthe in-vitro reaction of E. coli BL21/pET28-HDT1 extract withcaffeoyl-CoA donor substrate and L-DOPA acceptor substrate whichproduces a peak at approximately 10.245 minutes which is clovamide. FIG.4B is the reverse phase HPLC of the in-vitro reaction of E. coliBL21/pET28 (negative control) extract with caffeoyl-CoA donor substrateand L-DOPA substrate, producing no clovamide peak. FIG. 4C is thereverse phase HPLC of a clovamide standard demonstrating its retentiontime at approximately 10.247 minutes.

DETAILED DESCRIPTION OF THE INVENTION

The previously published data described above led to the hunt for thegene that encodes an enzyme that produces clovamide in red clover andwill produce clovamide in genetically altered alfalfa. This enzyme isdistinct from HMT (encoded by HCT2) (Sullivan, 2009, Plant Physiol.150:1866-1879). Using PCR, two novel red clover cDNAs (HDT1 (SEQ IDNO: 1) and HDT2 (SEQ ID NO: 3)) encoding HDT1 (SEQ ID NO: 2) and HDT2(SEQ ID NO: 4), respectively, are isolated and sequenced. See FIGS. 1A,1B, 2A, and 2B. The encoded enzymes,hydroxycinnamoyl-CoA:L-DOPA/tyrosine hydroxycinnamoyl transferase 1 andhydroxycinnamoyl-CoA:L-DOPA/tyrosine hydroxycinnamoyl transferase 2, areenzymatically active, contain 452 amino acids and differ by 5 aminoacids—at positions 140, 255, 285, 346, and 436. The cDNAs HDT1 and HDT2are each 1451 nucleotides long and differ by 12 nucleotides—at positions455, 458, 767, 801, 891, 920, 1034, 1074, 1244, 1292, 1344, and 1402.See FIG. 1A, FIG. 1B for cDNA sequence alignment, and FIG. 2 for aminoacid sequence alignment. When genetically altered alfalfa are generatedcontaining either HDT1 or HDT2, the genetically altered alfalfa producea protein having hydroxycinnamoyl-CoA:L-DOPA/tyrosine hydroxycinnamoyltransferase and can produce clovamide and/or other relatedhydroxycinnamoyl amides. Thus, these genetically altered alfalfa plantshave the reduced post-harvest degradation of proteins which improve thenutritional value of the genetically altered alfalfa.

The term “related hydroxycinnamoyl amides” refers to other compoundsproduced by HDT1 and/or HDT2 (a protein havinghydroxycinnamoyl-CoA:L-DOPA/tyrosine hydroxycinnamoyl transferase) andwhich reduce post-harvest degradation of proteins in plant containingthe related hydroxycinnamoyl amides (genetically altered alfalfa, redclover, etc.). These related hydroxycinnamoyl amides can be, but are notlimited to, N-caffeoyl-L-tyrosine,N-p-coumaroyl-L-3,4-dihydroxyphenylalanine (also calledN-p-coumaroyl-L-DOPA), and N-feruloyl-L-3,4-dihydroxyphenylalanine (alsocalled N-feruloyl-L-DOPA). Clovamide is also referred to asN-caffeoyl-L-3,4-dihydroxyphenylalanine or N-caffeoyl-L-DOPA.

Because this invention involves production of genetically altered plantsand involves recombinant DNA techniques, the following definitions areprovided to assist in describing this invention. The terms “isolated”,“purified”, or “biologically pure” as used herein, refer to materialthat is substantially or essentially free from components that normallyaccompany the material in its native state or when the material isproduced. In an exemplary embodiment, purity and homogeneity aredetermined using analytical chemistry techniques such as polyacrylamidegel electrophoresis or high performance liquid chromatography. A nucleicacid or particular bacteria that are the predominant species present ina preparation is substantially purified. In an exemplary embodiment, theterm “purified” denotes that a nucleic acid or protein that gives riseto essentially one band in an electrophoretic gel. Typically, isolatednucleic acids or proteins have a level of purity expressed as a range.The lower end of the range of purity for the component is about 60%,about 70% or about 80% and the upper end of the range of purity is about70%, about 80%, about 90% or more than about 90%.

The term “nucleic acid” as used herein, refers to a polymer ofribonucleotides or deoxyribonucleotides. Typically, “nucleic acid”polymers occur in either single- or double-stranded form, but are alsoknown to form structures comprising three or more strands. The term“nucleic acid” includes naturally occurring nucleic acid polymers aswell as nucleic acids comprising known nucleotide analogs or modifiedbackbone residues or linkages, which are synthetic, naturally occurring,and non-naturally occurring, which have similar binding properties asthe reference nucleic acid, and which are metabolized in a mannersimilar to the reference nucleotides. Exemplary analogs include, withoutlimitation, phosphorothioates, phosphoramidates, methyl phosphonates,chiral-methyl phosphonates, 2-O-methyl ribonucleotides, andpeptide-nucleic acids (PNAs). “DNA”, “RNA”, “polynucleotides”,“polynucleotide sequence”, “oligonucleotide”, “nucleotide”, “nucleicacid”, “nucleic acid molecule”, “nucleic acid sequence”, “nucleic acidfragment”, and “isolated nucleic acid fragment” are used interchangeablyherein.

For nucleic acids, sizes are given in either kilobases (kb) or basepairs (bp). Estimates are typically derived from agarose or acrylamidegel electrophoresis, from sequenced nucleic acids, or from published DNAsequences. For proteins, sizes are given in kilodaltons (kDa) or aminoacid residue numbers. Proteins sizes are estimated from gelelectrophoresis, from sequenced proteins, from derived amino acidsequences, or from published protein sequences.

Unless otherwise indicated, a particular nucleic acid sequence alsoimplicitly encompasses conservatively modified variants thereof (e.g.,degenerate codon substitutions), the complementary (or complement)sequence, and the reverse complement sequence, as well as the sequenceexplicitly indicated. Specifically, degenerate codon substitutions maybe achieved by generating sequences in which the third position of oneor more selected (or all) codons is substituted with mixed-base and/ordeoxyinosine residues (see e.g., Batzer et al., Nucleic Acid Res.19:5081 (1991); Ohtsuka et al., J. Biol. Chem. 260:2605-2608 (1985); andRossolini et al., Mol. Cell. Probes 8:91-98(1994)). Because the aminoacid sequences of SEQ ID NO: 2 and SEQ ID NO: 4 are described herein,one can chemically synthesize a polynucleotide which encodes theseenzymes. Because of the degeneracy of nucleic acid codons, one can usevarious different polynucleotides to encode identical proteins. Table 1,infra, contains information about which nucleic acid codons encode whichamino acids.

TABLE 1 Amino acid Nucleic acid codons Ala/A GCT, GCC, GCA, GCG Arg/RCGT, CGC, CGA, CGG, AGA, AGG Asn/N AAT, AAC Asp/D GAT, GAC Cys/CTGT, TGC Gln/Q CAA, CAG Glu/E GAA, GAG Gly/G GGT, GGC, GGA, GGG His/HCAT, CAC Ile/I ATT, ATC, ATA Leu/L TTA, TTG, CTT, CTC, CTA, CTG Lys/KAAA, AAG Met/M ATG Phe/F TTT, TTC Pro/P CCT, CCC, CCA, CCG Ser/STCT, TCC, TCA, TCG, AGT, AGC Thr/T ACT, ACC, ACA, ACG Trp/W TGG Tyr/YTAT, TAC Val/V GTT, GTC, GTA, GTG

In addition to the degenerate nature of the nucleotide codons whichencode amino acids, alterations in a polynucleotide that result in theproduction of a chemically equivalent amino acid at a given site, but donot affect the functional properties of the encoded protein, are wellknown in the art. “Conservative amino acid substitutions” are thosesubstitutions that are predicted to interfere least with the propertiesof the reference protein. In other words, conservative amino acidsubstitutions substantially conserve the structure and the function ofthe reference protein. Thus, a codon for the amino acid alanine, ahydrophobic amino acid, may be substituted by a codon encoding anotherless hydrophobic residue, such as glycine, or a more hydrophobicresidue, such as valine, leucine, or isoleucine. Similarly, changeswhich result in substitution of one negatively charged residue foranother, such as aspartic acid for glutamic acid, or one positivelycharged residue for another, such as lysine for arginine or histidine,can also be expected to produce a functionally equivalent protein orpolypeptide. Table 2 provides a list of exemplary conservative aminoacid substitutions. Conservative amino acid substitutions generallymaintain (a) the structure of the protein backbone in the area of thesubstitution, for example, as a beta sheet or alpha helicalconformation, (b) the charge or hydrophobicity of the molecule at thesite of the substitution, and/or (c) the bulk of the side chain.

TABLE 2 Amino Acid Conservative Substitute Ala Gly, Ser Arg His, Lys AsnAsp, Gln, His Asp Asn, Glu Cys Ala, Ser Gln Asn, Glu, His Glu Asp, Gln,His Gly Ala His Asn, Arg, Gln, Glu Ile Leu, Val Leu Ile, Val Lys Arg,Gln, Glu Met Ile, Leu Phe His, Leu, Met, Trp, Tyr Ser Cys, Thr Thr Ser,Val Trp Phe, Tyr Tyr His, Phe, Trp Val Ile, Leu, Thr

Oligonucleotides and polynucleotides that are not commercially availablecan be chemically synthesized e.g., according to the solid phasephosphoramidite triester method first described by Beaucage andCaruthers, Tetrahedron Letts. 22:1859-1862 (1981), or using an automatedsynthesizer, as described in Van Devanter et al., Nucleic Acids Res.12:6159-6168 (1984). Other methods for synthesizing oligonucleotides andpolynucleotides are known in the art. Purification of oligonucleotidesis by either native acrylamide gel electrophoresis or by anion-exchangeHPLC as described in Pearson & Reanier, J. Chrom. 255:137-149 (1983).

The terms “identical” or percent “identity”, in the context of two ormore nucleic acids or polypeptide sequences, refer to two or moresequences or subsequences that are the same or have a specifiedpercentage of amino acid residues or nucleotides that are the same(e.g., 80%, 85% identity, 90% identity, 99%, or 100% identity), whencompared and aligned for maximum correspondence over a designated regionas measured using a sequence comparison algorithm or by manual alignmentand visual inspection.

The phrase “high percent identical” or “high percent identity”, in thecontext of two polynucleotides or polypeptides, refers to two or moresequences or subsequences that have at least about 80%, identity, atleast about 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% nucleotide or amino acidresidue identity, when compared and aligned for maximum correspondence,as measured using a sequence comparison algorithm or by visualinspection. In an exemplary embodiment, a high percent identity existsover a region of the sequences that is at least about 50 residues inlength. In another exemplary embodiment, a high percent identity existsover a region of the sequences that is at least about 100 residues inlength. In still another exemplary embodiment, a high percent identityexists over a region of the sequences that is at least about 150residues or more in length. In one exemplary embodiment, the sequencesare high percent identical over the entire length of the nucleic acid orprotein sequence.

For sequence comparison, typically one sequence acts as a referencesequence, to which test sequences are compared. When using a sequencecomparison algorithm, test and reference sequences are entered into acomputer, subsequence coordinates are designated, if necessary, andsequence algorithm program parameters are designated. Default programparameters can be used, or alternative parameters can be designated. Thesequence comparison algorithm then calculates the percent sequenceidentities for the test sequences relative to the reference sequence,based on the program parameters. Methods of alignment of sequences forcomparison are well-known in the art. Optimal alignment of sequences forcomparison can be conducted, e.g., by the local homology algorithm ofSmith & Waterman, Adv. Appl. Math. 2:482 (1981), by the homologyalignment algorithm of Needleman & Wunsch, J. Mol. Biol. 48:443 (1970),by the search for similarity method of Pearson & Lipman, Proc. Natl.Acad. Sci. USA 85:2444 (1988), by computerized implementations of thesealgorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin GeneticsSoftware Package, Genetics Computer Group, 575 Science Dr., Madison,Wis.), or by manual alignment and visual inspection (see, e.g., Ausubelet al. (eds.), Current Protocols in Molecular Biology, 1995 supplement).

The term “recombinant” when used with reference, e.g., to a cell, ornucleic acid, protein, or vector, indicates that the cell, organism,nucleic acid, protein or vector, has been modified by the introductionof a heterologous nucleic acid or protein or the alteration of a nativenucleic acid or protein, or that the cell is derived from a cell somodified. Thus, for example, recombinant cells may express genes thatare not found within the native (non-recombinant or wild-type) form ofthe cell or express native genes that are otherwise abnormallyexpressed—over-expressed, under-expressed or not expressed at all.

The terms “transgenic”, “transformed”, “transformation”, and“transfection” are similar in meaning to “recombinant”.“Transformation”, “transgenic”, and “transfection” refer to the transferof a polynucleotide into the genome of a host organism or into a cell.Such a transfer of polynucleotides can result in genetically stableinheritance of the polynucleotides or in the polynucleotides remainingextra-chromosomally (not integrated into the chromosome of the cell).Genetically stable inheritance may potentially require the transgenicorganism or cell to be subjected for a period of time to one or moreconditions which require the transcription of some or all of thetransferred polynucleotide in order for the transgenic organism or cellto live and/or grow. Polynucleotides that are transformed into a cellbut are not integrated into the host's chromosome remain as anexpression vector within the cell. One may need to grow the cell undercertain growth or environmental conditions in order for the expressionvector to remain in the cell or the cell's progeny. Further, forexpression to occur the organism or cell may need to be kept undercertain conditions. Host organisms or cells containing the recombinantpolynucleotide can be referred to as “transgenic” or “transformed”organisms or cells or simply as “transformants”, as well as recombinantorganisms or cells.

A genetically altered organism is any organism with any change to itsgenetic material, whether in the nucleus or cytoplasm (organelle). Assuch, a genetically altered organism can be a recombinant or transformedorganism. A genetically altered organism can also be an organism thatwas subjected to one or more mutagens or the progeny of an organism thatwas subjected to one or more mutagens and has changes in its DNA causedby the one or more mutagens, as compared to the wild-type organism (i.e,organism not subjected to the mutagens). Also, an organism that has beenbred to incorporate a mutation into its genetic material is agenetically altered organism. For the purposes of this invention, theorganism is a plant.

The term “vector” refers to some means by which DNA, RNA, a protein, orpolypeptide can be introduced into a host. The polynucleotides, protein,and polypeptide which are to be introduced into a host can betherapeutic or prophylactic in nature; can encode or be an antigen; canbe regulatory in nature; etc. There are various types of vectorsincluding virus, plasmid, bacteriophages, cosmids, and bacteria.

An expression vector is nucleic acid capable of replicating in aselected host cell or organism. An expression vector can replicate as anautonomous structure, or alternatively can integrate, in whole or inpart, into the host cell chromosomes or the nucleic acids of anorganelle, or it is used as a shuttle for delivering foreign DNA tocells, and thus replicate along with the host cell genome. Thus, anexpression vector are polynucleotides capable of replicating in aselected host cell, organelle, or organism, e.g., a plasmid, virus,artificial chromosome, nucleic acid fragment, and for which certaingenes on the expression vector (including genes of interest) aretranscribed and translated into a polypeptide or protein within thecell, organelle or organism; or any suitable construct known in the art,which comprises an “expression cassette”. In contrast, as described inthe examples herein, a “cassette” is a polynucleotide containing asection of an expression vector of this invention. The use of thecassettes assists in the assembly of the expression vectors. Anexpression vector is a replicon, such as plasmid, phage, virus, chimericvirus, or cosmid, and which contains the expression control sequence(s)operably linked to the desired polynucleotide sequence.

A polynucleotide sequence is operably linked to an expression controlsequence(s) (e.g., a promoter and, optionally, an enhancer) when theexpression control sequence controls and regulates the transcriptionand/or translation of that polynucleotide sequence.

As used herein, the term “promoter” refers to a polynucleotide that inits native state is located upstream or 5′ to a translational startcodon of an open reading frame (or protein-coding region) and that isinvolved in recognition and binding of RNA polymerase and other proteins(trans-acting transcription factors) to initiate transcription. A “plantpromoter” is a native or non-native promoter that is functional in plantcells. The promoters that predominately function in particular cellsand/or tissue are considered “tissue-specific promoters”. A plantpromoter can be used as a 5′ regulatory element for modulatingexpression of a particular desired polynucleotide (heterologouspolynucleotide) operably linked thereto. When operably linked to atranscribeable polynucleotide, a promoter typically causes thetranscribable polynucleotide to be transcribed in a manner that issimilar to that of which the promoter is normally associated. Thistranscribeable polynucleotide can be heterologous to the promoter, orheterologous to the organism into which the cassette will betransfected, or both.

A heterologous polynucleotide sequence is operably linked to one or moretranscription regulatory elements (e.g., promoter, terminator and,optionally, enhancer) such that the transcription regulatory elementscontrol and regulate the transcription and/or translation of thatheterologous polynucleotide sequence. A cassette can have theheterologous polynucleotide operably linked to one or more transcriptionregulatory elements. As used herein, the term “operably linked” refersto a first polynucleotide, such as a promoter, connected with a secondtranscribable polynucleotide, such as a gene of interest, where thepolynucleotides are arranged such that the first polynucleotide affectsthe transcription of the second polynucleotide. In some embodiments, thetwo polynucleotide molecules are part of a single contiguouspolynucleotide. In other embodiments, the two polynucleotides areadjacent. For example, a promoter is operably linked to a gene ofinterest if the promoter regulates or mediates transcription of the geneof interest in a cell. Similarly a terminator is operably linked to thepolynucleotide of interest if the terminator regulates or mediatestranscription of the polynucleotide of interest, and in particular, thetermination of transcription. Constructs of the present invention wouldtypically contain a promoter operably linked to a transcribablepolynucleotide operably linked to a terminator.

Exemplary heterologous polynucleotide for incorporation into constructsof the present invention include, for example, desired polynucleotidesfrom a species other than the target plant's species, or even desiredpolynucleotides that originate with or are present in the same plantspecies, but are incorporated into the genetically altered plant cellsby genetic engineering methods rather than classical reproduction orbreeding techniques or by a combination of genetic engineering methodsfollowed by breeding techniques. Heterologous polynucleotides refer toany polynucleotide molecule that is introduced into a recipient cell andis transcribed at levels that differ from the wild-type cell. Aheterologous polynucleotide can include a polynucleotide that is alreadypresent in the plant cell, polynucleotide from another plant,polynucleotide from a different organism, or a polynucleotide generatedexternally, such as a polynucleotide containing an antisense message ofa gene, or a polynucleotide encoding an artificial or modified versionof a gene.

Transformation and generation of genetically altered monocotyledonousand dicotyledonous plant cells is well known in the art. See, e.g.,Weising, et al., Ann. Rev. Genet. 22:421-477 (1988); U.S. Pat. No.5,679,558; Agrobacterium Protocols, ed: Gartland, Humana Press Inc.(1995); and Wang, et al. Acta Hort. 461:401-408 (1998). The choice ofmethod varies with the type of plant to be transformed, the particularapplication and/or the desired result. The appropriate transformationtechnique is readily chosen by the skilled practitioner.

Exemplary transformation/transfection methods available to those skilledin the art include, but are not limited to: direct uptake of foreign DNAconstructs (see, e.g., EP 295959); techniques of electroporation (see,e.g., Fromm et al., Nature 319:791 (1986)); and high-velocity ballisticbombardment with metal particles coated with the nucleic acid constructs(see, e.g., Kline, et al., Nature 327:70 (1987) and U.S. Pat. No.4,945,050). Specific methods to transform heterologous genes intocommercially important crops (to make genetically altered plants) arepublished for rapeseed (De Block, et al., Plant Physiol. 91:694-701(1989)); sunflower (Everett, et al., Bio/Technology 5:1201 (1987));soybean (McCabe, et al., Bio/Technology 6:923 (1988), Hinchee, et al.,Bio/Technology 6:915 (1988), Chee, et al., Plant Physiol. 91:1212-1218(1989), and Christou, et al., Proc. Natl. Acad. Sci USA 86:7500-7504(1989)); rice (Hiei, et al., Plant J. 6:271-282 (1994)), and corn(Gordon-Kamm, et al., Plant Cell 2:603-618 (1990), and Fromm, et al.,Biotechnology 8:833-839 (1990)). Other known methods are disclosed inU.S. Pat. Nos. 5,597,945; 5,589,615; 5,750,871; 5,268,526; 5,262,316;and 5,569,831.

One exemplary method includes employing Agrobacterium tumefaciens orAgrobacterium rhizogenes as the transforming agent to transferheterologous DNA into the plant. Agrobacterium tumefaciens-meditatedtransformation techniques are well described in the scientificliterature. See, e.g., Horsch, et al. Science 233:496-498 (1984), andFraley, et al. Proc. Natl. Acad. Sci. USA 80:4803 (1983). Typically, aplant cell, an explant, a meristem or a seed is infected withAgrobacterium tumefaciens transformed with the expressionvector/construct which contains a promoter operably linked to theheterologous nucleic acid. Under appropriate conditions known in theart, the transformed plant cells are grown to form shoots, roots, anddevelop further into genetically altered plants. In some embodiments,the heterologous nucleic acid can be introduced into plant cells, bymeans of the Ti plasmid of Agrobacterium tumefaciens. The Ti plasmid istransmitted to plant cells upon infection by Agrobacterium tumefaciens,and is stably integrated into the plant genome. See, e.g., Horsch, etal. (1984), and Fraley, et al. (1983).

Transformed plant cells which are derived by any of the abovetransformation techniques can be cultured to regenerate a whole plantwhich possesses the desired transformed phenotype. Such regenerationtechniques rely on manipulation of certain phytohormones in a tissueculture growth medium, typically relying on a biocide and/or herbicidemarker which has been introduced together with the desired nucleotidesequences. Plant regeneration from cultured protoplasts is described inEvans et al., Protoplasts Isolation and Culture, in Handbook of PlantCell Culture, pp. 124-176, MacMillan Publishing Company, New York, 1983;and Binding, Regeneration of Plants, in Plant Protoplasts, pp. 21-73,CRC Press, Boca Raton, 1985. Regeneration can also be obtained fromplant callus, explants, organs, or parts thereof. Such regenerationtechniques are described generally in Klee, et al., Ann. Rev. of PlantPhys. 38:467-486 (1987).

Once a genetically altered diploid plant has been generated, one canbreed it with a wild-type plant and screen for heterozygous F1generation diploid plants containing the genetic change present in theparent genetically altered plant. Then F2 generation diploid plants canbe generated which are homozygous for the genetic alteration for diploidspecies. These heterozygous F1 generation plants and homozygous F2plants, progeny of the original genetically altered plant, areconsidered genetically altered plants, having the altered genomicmaterial from the genetically altered parent plant. Alfalfa isautotetraploid; thus among F2 plants, some would have two copies of thegene. Generation of F3, and possibly F4 crosses, are required to producehomozygous autoploidy plants.

After one obtains a genetically altered plant expressing theheterologous protein, one can efficiently breed the genetically alteredplant with other plants containing desired traits. One can use molecularmarkers (i.e., polynucleotide probes) based on the sequence of theheterologous protein as described above to determine which offspring ofcrosses between the genetically altered plant and the other plant havethe polynucleotide encoding the chimeric protein. This process is knownas Marker Assisted Rapid Trait Introgression (MARTI). Briefly, MARTIinvolves (1) crossing the genetically altered plant with a plant linehaving desired phenotype/genotype (“elite parent”) for introgression toobtain F1 offspring. The F1 generation is heterozygous for chimericprotein trait. (2) Next, an F1 plant is be backcrossed to the eliteparent, producing BC1F1 which genetically produces 50% wild-type and 50%heterozygote chimeric protein. (3) PCR using the polynucleotide probe isperformed to select the heterozygote genetically altered plantscontaining polynucleotide encoding the chimeric protein. (4) Selectedheterozygotes are then backcrossed to the elite parent to performfurther introgression. (5) This process of MARTI is performed foranother four cycles. (6) Next, the heterozygote genetically alteredplant is self-pollinated by bagging to produce BC6F2 generation. TheBC6F2 generation produces a phenotypic segregation ratio of 3 wild-typeparent plants to 1 chimeric protein genetically altered plant. (7) Oneselects genetically altered plants expressing the protein of interest atthe BC6F2 generation at the seedling stage using PCR with thepolynucleotide probe and can optionally be combined with phenotypicselection at maturity. These cycles of crossing and selection can beachieved in a span of 2 to 2.5 years (depending on the plant), ascompared to many more years for conventional backcrossing introgressionmethod now in use. Thus, the application of MARTI using PCR with apolynucleotide probe significantly reduces the time to introgress thechimeric protein genetic alteration into elite lines for producingcommercial hybrids. The final product is an inbred plant line almostidentical (99%) to the original elite in-bred parent plant that is thehomozygous for the polynucleotide encoding the chimeric protein.Alternatively, one can apply PCR to one of the methods of breedingalfalfa (described above) to identify those genetically altered alfalfaplants offspring that contain the desired genotype and phenotype. Suchan approach is still referred to as MARTI, and introgression stillrefers to the transferring of a desired genotype/phenotype to theprogeny of a cross between alfalfa containing differentgenotypes/phenotypes.

This invention utilizes routine techniques in the field of molecularbiology. Basic texts disclosing the general methods of use in thisinvention include Green and Sambrook, 4th ed. 2012, Cold Spring HarborLaboratory; Kriegler, Gene Transfer and Expression: A Laboratory Manual(1993); and Ausubel et al., eds., Current Protocols in MolecularBiology, 1994—current, John Wiley & Sons. Unless otherwise noted,technical terms are used according to conventional usage. Definitions ofcommon terms in molecular biology maybe found in e.g., Benjamin Lewin,Genes IX, published by Oxford University Press, 2007 (ISBN 0763740632);Krebs, et al. (eds.), The Encyclopedia of Molecular Biology, publishedby Blackwell Science Ltd., 1994 (ISBN 0-632-02182-9); and Robert A.Meyers (ed.), Molecular Biology and Biotechnology: a Comprehensive DeskReference, published by VCH Publishers, Inc., 1995 (ISBN 1-56081-569-8).

The term “plant” includes whole plants, plant organs, progeny of wholeplants or plant organs, embryos, somatic embryos, embryo-likestructures, protocorms, protocorm-like bodies (PLBs), and suspensions ofplant cells. Plant organs comprise, e.g., shoot vegetativeorgans/structures (e.g., leaves, stems and tubers), roots, flowers andfloral organs/structures (e.g., bracts, sepals, petals, stamens,carpels, anthers and ovules), seed (including embryo, endosperm, andseed coat) and fruit (the mature ovary), plant tissue (e.g., vasculartissue, ground tissue, and the like) and cells (e.g., guard cells, eggcells, trichomes and the like). The class of plants that can be used inthe method of the invention is generally as broad as the class of higherand lower plants amenable to the molecular biology and plant breedingtechniques described herein, specifically angiosperms (monocotyledonous(monocots) and dicotyledonous (dicots) plants). It includes plants of avariety of ploidy levels, including aneuploid, polyploid, diploid,haploid and hemizygous. The genetically altered plants described hereinalfalfa.

The terms “approximately” and “about” refer to a quantity, level, valueor amount that varies by as much as 30%, or in another embodiment by asmuch as 20%, and in a third embodiment by as much as 10% to a referencequantity, level, value or amount. As used herein, the singular form “a”,“an”, and “the” include plural references unless the context clearlydictates otherwise. For example, the term “a bacterium” includes both asingle bacterium and a plurality of bacteria. All references mentionedherein are incorporated by reference.

Having now generally described this invention, the same will be betterunderstood by reference to certain specific examples and theaccompanying drawings, which are included herein only to furtherillustrate the invention and are not intended to limit the scope of theinvention as defined by the claims. The examples and drawings describeat least one, but not all embodiments, of the inventions claimed.Indeed, these inventions may be embodied in many different forms andshould not be construed as limited to the embodiments set forth herein;rather, these embodiments are provided so that this disclosure willsatisfy applicable legal requirements.

Example 1. Hydroxycinnamoyl-CoA:L-DOPA Hydroxycinnamoyl TransferaseActivity in Red Clover Tissues

For experiments described in this and other examples herein with redclover (Trifolium pratense), a highly regenerable genotype (designatedNRC7) derived from a population of NewRC germplasm (Smith andQuesenberry, 1995, Crop Sci. 35:295-295) was used. For geneticallyaltered alfalfa (Medicago sativa), a highly regenerable clone ofRegen-SY germplasm (Bingham, 1991, Crop Sci. 31:1098) was used for allexamples. A collection of NRC7 red clover plants silenced for red cloverhydroxycinnamoyl-CoA:malate hydroxycinnamoyl transferase (HMT, encodedby red clover HCT2) was generated as described by Sullivan andZarnowski, 2011, using the RNAi construct described therein whichcontains a hairpin RNA corresponding to the region between nucleotides481 and 1224 of GenBank sequence EU861219.

Further, for all experiments described herein, trans-p-coumaroyl-,-caffeoyl-, and -feruloyl-CoA thiolesters were prepared usingrecombinant A. thaliana 4CL1 protein (Lee, et al., 1995 Plant Mol. Biol.28:871-884) produced in Escherichia coli using the pET30 expressionvector (Novagen, Madison, Wis.) and quantified as detailed in Sullivan,2009).

To assess the accumulation of hydroxycinnamoyl compounds in plants,tissue samples were ground in liquid nitrogen in a mortar and pestle, orfor small samples in a 2 mL screw cap tube with two 4 mm glass beadsusing a Mini-BeadBeater (Biospec Products, Bartlesville, Okla.). Theground frozen tissue was extracted at room temperature with 10 mL/g 100mM HCl, 50 mM ascorbic acid. Extracts were filtered through Miracloth(Calbiochem, Billerica, Mass.) or glass wool then centrifuged at20,000×g at room temperature. 1 mL of the resulting supernatant wasapplied to a 1 mL ENVI-18 solid phase extraction column (Supelco, St.Louis, Mo., USA) pre-equilibrated with 3×1 mL of methanol and 3×1 mL0.1% acetic acid in water (pH adjusted to 2.5 with HCl). The column waswashed with 3×1 mL 0.1% acetic acid in water (pH adjusted to 2.5 withHCl) and eluted with 1 mL methanol.

The eluate is analyzed for hydroxycinnamates and other phenolics byHPLC. The eluents were analyzed on a Shim-Pack XR-ODS II (C-18) 120 Åcolumn (Shimadzu Scientific Instruments North America, Columbia, Md.,USA; 100×2.0 mm×2.2 micron) using a two solvent system [Solvent A:deionized water with 0.1% (v/v) formic acid, Solvent B: acetonitrile] ata flow rate of 0.5 mL/min. The HPLC conditions were 5 min isocratic 2%Solvent B, 10 min gradient to 30% Solvent B, 3 min gradient to 100%Solvent B, 5 min isocratic 100% Solvent B, 0.5 min gradient to 2%Solvent B and 3.5 min isocratic re-equilibration at 2% Solvent B.Compound elution was monitored (250 to 500 nm) with a UV/visiblephotodiode array detector (PDA). When peaks were quantified, purchasedclovamide or free hydroxycinnamic acids were used as standards (Nielsen,et al., 1984, Phytochem 23:1741-1743; Sullivan and Zeller, 2012). Insome cases, elution was also monitored by mass spectrometry using aMS2020 mass spectrometer (MS) (Shimadzu Scientific Instruments NorthAmerica) using a dual ion source (electrospray and atmospheric pressurechemical ionization) with data collection in both positive and negativeion modes. MS data was collected between 2.0 and 16.0 min of the HPLCrun, scanning for m/z between 50 and 500 u at 7500 u/sec, with detectorvoltage of 1.3 kV, nebulizing gas flow of 1.5 L/min, drying gas flow of10 L/min, desolvation line and heat block temperatures of 250° C.

Plant tissue extracts were prepared to assess hydroxycinnamoyl-CoAhydroxycinnamoyl transferase activity. Tissue was powdered in liquidnitrogen using a mortar and pestle or for small samples in a 2 mL screwcap tube with two 4 mm glass beads using a Mini-BeadBeater (BiospecProducts, Bartlesville, Okla.). The frozen powdered tissue was added to1 to 2 mL/g extraction buffer containing 100 mM Na phosphate (pH 7.5),100 mM ascorbic acid (pH adjusted to 7.5 with NaOH), and 1% (v/v)protease inhibitor cocktail (P-9599, Sigma, St. Louis, Mo.). The frozen,powdered tissue and buffer were thoroughly mixed by stirring or vortexmixing (depending on amount of tissue and volume) until the mixturethawed and reached a temperature of 6 to 8° C. The slurry was filteredthrough a layer of Miracloth (Calbiochem, Billerica, Mass.) on top of adouble layer of cheesecloth, as much liquid as possible was squeezedout, and the filtrate collected on ice. The filtrate was divided amongmicrocentrifuge tubes and centrifuged at 17,000×g at 4° C. for 5 min.The supernatant was removed to fresh microcentrifuge tubes, thecentrifugation repeated, and the supernatant retained. Supernatants(typically 30% of the packed column volume) were applied to previouslyprepared spin columns (1-10 mL syringes packed with Sephadex G-25Superfine [GE Healthcare, Uppsala, Sweden] equilibrated with 100 mM Naphosphate [pH 7.5 or as specified for individual experiments], andcentrifuged for 1 min at 200×g prior to sample application) to removelow molecular weight compounds, and in some cases, to change the pH ofthe extract. Following supernatant application, the columns werecentrifuged for 2 min at 200×g and the flow through (desalted proteinextract) retained. Following addition of fresh protease inhibitorcocktail (to 0.5% [v/v]), extracts were divided into 150 to 200 μLaliquots, flash frozen in liquid nitrogen, and stored at −80° C. untilneeded. In the case of pH adjustment by the spin column procedure, pHwas confirmed by spotting a small amount of extract on pH indicatorpaper. Protein content of the extracts was determined using Bio-RadProtein Assay (Bio-Rad Laboratories, Hercules, Calif.) using bovineserum albumin as the standard.

In-vitro reactions for hydroxycinnamoyl-CoA transferase activitycontained 100 mM sodium phosphate buffer (pH 7.5), 25 to 50 mMascorbate, 1 to 2 mM p-coumaroyl-, caffeoyl-, or feruloyl-CoA donorsubstrate, 1 to 6 mM acceptor substrate (L-DOPA, tyrosine, shikimic, ormalic acid), and enzyme source (e.g. leaf extracts as prepared above orsoluble E. coli extract as described below). Reactions were incubated at30° C. for up to 3 hours then stopped by the addition of ⅕ volume of 10%formic acid. Precipitated protein was removed by centrifugation(17,000×g for 5 min at room temperature). The supernatant is analyzedfor reaction products by HPLC with PDA and sometimes MS detection asdescribed above.

Using this approach, a previously undescribedhydroxycinnamoyl-CoA:L-DOPA hydroxycinnamoyl transferase activity (HDT)was detected in unexpanded red clover leaves (15 pkat/mg protein forcaffeoyl-CoA donor and L-DOPA acceptor substrates), although no HDTactivity could be detected in mature red clover leaves. Among plantstransformed with a hairpin RNA RNAi gene silencing construct for HCT2(which encodes HMT), three independent transformants whose average levelof phaselic acid was reduced greater than 100-fold relative to wild typecontrols also showed greater than 10-fold reductions in clovamide levelsrelative to wild type controls. In these plants, HMT activity (thetarget of the silencing transgene) in unexpanded leaves was reduced toundetectable levels, and HDT activity was reduced by nearly 20-fold.Previous experiments with HCT2 gene product produced in E. coliindicated that it does not have HDT activity, however (Sullivan, 2009).Based on these results and the data previously published, it washypothesized that the gene encoding HDT is expressed in unexpandedleaves but not mature leaves. As such, a PCR approach to isolation andidentification of a cDNA corresponding to the red clover gene encodingHDT activity was taken.

Example 2. Cloning of Two cDNAs Encoding Hydroxycinnamoyl-CoA:L-DOPAHydroxycinnamoyl Transferse (HDT1 and HDT2)

Red clover total RNA was prepared from plant tissues using the RNeasyPlant Mini Kit (Qiagen, Valencia, Calif.) according to themanufacturer's recommended protocol. Oligo dT-primed cDNA was preparedusing Superscript III reverse transcriptase (Invitrogen, Carlsbad,Calif.) according to the manufacturer's recommended protocol from DNaseI-treated total RNA. Plasmid DNA was prepared using the QIAprep SpinMiniprep Kit (Qiagen, Valencia, Calif.) according to the manufacturer'srecommended protocol. DNA sequence was determined by Sanger cyclesequencing via reactions using Big Dye v3.1 (Applied Biosystems, FosterCity, Calif.). Sequencing reactions were analyzed on ABI 3730xl DNAAnalyzers by the University of Wisconsin Biotechnology Center (Madison,Wis.). Sequence analyses were carried out using the Lasergene Version 8or higher (DNAStar, Madison, Wis.), and BLAST® programs using theNational Center for Biotechnology Information (NCBI, ncbi.nlm.nih.gov)web site.

A nested PCR strategy using degenerate primers based on conservedregions of previously cloned red clover hydroxycinnamoyl-CoA:malate andhydroxycinnamoyl-CoA:shikimate hydroxycinnamoyl transferases (GenBankaccessions EU861219 and EU861218, respectively) as well as twouncharacterized Phaseolus vulgaris putative hydroxycinnamoyl-CoAhydroxycinnamoyl transferases (GenBank Accessions XM_007146186 andXM_007146336) was used to obtain a DNA fragment corresponding to HDT.The PCR was carried out using Phusion® DNA polymerase (New EnglandBiolabs, Ipswich, Mass.). The first round PCR reaction (50 μL) contained1× Phusion® HF Buffer, 200 μM dNTP, cDNA equivalent to 100 ng total RNAfrom unexpanded red clover leaves (prepared as described above), 1 unitPhusion® DNA polymerase, 1 μM each primers ms809 (SEQ ID NO: 5) andms815 (SEQ ID NO: 6) (Table 3, infra). The PCR reaction was incubatedfor 30 sec at 98° C. in the preheated block of a thermocycler. Thisincubation was followed by 35 cycles of 98° C. for 10 sec(denaturation), 55° C. for 20 sec (annealing), and 72° C. for 30 sec(extension) followed by a final 5 min extension at 72° C. 20 μL of thePCR reaction was resolved via electrophoresis on a 1.0% agarose gelusing standard techniques (Sambrook, et al., 2012). An approximately1000 bp DNA fragment was excised from the gel and purified using QiaEx®resin (silica-gel particles) (Qiagen Inc., Germantown, Md.) according tothe manufacturer's recommended procedure.

A second round (nested) PCR reaction (25 μL) contained 1× Phusion® HFBuffer, 200 μM dNTP, gel purified first round PCR product equivalent to0.01 μL of the first round reaction, 0.5 units Phusion® DNA polymerase,0.5 μM each primers ms867 (SEQ ID NO: 7) and ms870 (SEQ ID NO: 8) (Table3, infra). The PCR reaction was incubated for 30 sec at 98° C. in thepreheated block of a thermocycler. This incubation was followed by 35cycles of 98° C. for 10 sec (denaturation), 49° C. for 20 sec(annealing), and 72° C. for 30 sec (extension) followed by a final 5 minextension at 72° C. 20 μL of the PCR reaction was resolved viaelectrophoresis on a 1.0% agarose gel using standard techniques. Anapproximately 700 bp DNA fragment was excised from the gel and purifiedusing QiaEx® resin (silica-gel particles) (Qiagen Inc., Germantown, Md.)according to the manufacturer's recommended procedure. The DNA fragmentwas cloned into pGEM T-Easy (Promega Corp., Madison, Wis.) according tothe manufacturer's recommended protocol.

Sequence of the resulting DNA fragment was used to design primers for 5′and 3′ RACE (rapid amplification of cDNA ends). 5′ and 3′ RACE werecarried out using the SMARTer® RACE cDNA Amplification Kit (Catalog#634923, Clontech Laboratories, Mountain View, Calif.) using total RNAfrom unexpanded red clover leaves and ms881 (SEQ ID NO: 9) and ms882(SEQ ID NO: 10) (see Table 3, infra) as the gene specific primers for 5′and 3′ RACE, respectively. The resulting 5′ and 3′ RACE products weregel purified as described above and cloned into pGEM T-Easy (PromegaCorp., Madison, Wis.) as described above. Sequencing of the resultingfragments was used to design primers ms884 (SEQ ID NO: 11) and ms885(SEQ ID NO: 12) (see Table 3, infra) for end to end PCR. Threeindependent end to end PCR reactions (25 μL each) contained 1× Phusion®HF Buffer, 200 μM dNTP, first strand 5′ RACE cDNA (equivalent to 17 ngtotal RNA), 0.5 units Phusion® DNA polymerase, 0.5 μM each primers ms884(SEQ ID NO: 11) and ms885 (SEQ ID NO: 12). The resulting DNA fragmentswere cloned into pGEM T-Easy (Promega Corp., Madison, Wis.) according tothe manufacturer's recommended protocol and several clones from each PCRreactions were sequenced.

Degenerate oligonucleotide PCR primers were designed based on severalconserved regions of red clover HCT2, red clover HCT1 (encodinghydroxycinnamoyl-CoA:shikimate hydroxycinnamoyl transferase, GenbankEU861218), and two other uncharacterized putative hydroxycinnamoyltransferase genes (Genbank XM_007146186 and XM_007146336) from Phaseolusvulgaris, another legume species. These primers were used in nested PCRreactions to generate an approximately 700 bp DNA fragment. Sequenceanalysis of the fragment revealed that it was distinct from the redclover HCT2 sequence (80% identity) and allowed primers to be designedfor 5′ and 3′ RACE (rapid amplification of mRNA ends) as describedabove. The resulting RACE products were sequenced and used to designprimers for end-to-end PCR to generate full-length clones correspondingto the putative HDT cDNA. For end-to-end PCR, a high fidelityproofreading thermostable DNA polymerase was used, and clones wereisolated and sequenced from three independent PCR reactions allowingauthentic alleles of the putative HDT gene to be distinguished from PCRerrors (true alleles or closely related members of a multigene familywould be expected to be represented in all three independent PCRreactions, whereas this would be unlikely for nucleotide changesresulting from DNA polymerase misincorporation).

Using this approach, two distinct 1451 bp cDNAs (SEQ ID NO: 1 (HDT1) andSEQ ID NO: 3 (HDT2), respectively) were identified. The cDNAs are >99%identical and are predicted to encode 452 amino acid proteins (SEQ IDNO: 2 (HDT1) and SEQ ID NO: 4 (HDT2), respectively) that are >98%identical. For red clover sequences in GenBank, the following highsimilarity (>90% identity over >50 bp) matches to HDT1 were found:embILN846355.1, unannotated genome assembly (97% identity over 847 nt,from Ser. No. 15/778,318 to Ser. No. 15/777,475, 95% identity over 400nt from Ser. No. 15/779,814 to Ser. No. 15/779,415); gblASHM01031115.1,unannotated whole genome shotgun sequence (99% identity over 530 bp fromnt 2103 to 1574, 99% identity from 484 to 1); gblASHM01105919.1,unannotated whole genome shotgun sequence (99% identity over 484 nt from484 to 1); embICVOM01002144.1, unannotated whole genome shotgut sequence(99% identity over 455 nt from 6373 to 6827, 99% identity over 251 ntfrom 7003 to 7253, 99% identity over 245 bp from 5443 to 5687, 94%identity over 79 bases from 6273 to 6351); gblASHM01081496.1,unannotated whole genome shotgun sequence (99% identity over 360 bp from1011 to 652); embICVOM01021691.1, unannotated whole genome shotgunsequence (99% identity over 223 bp, from 1 to 223); gblASHM01023500.1,unanotated whole genome shotgun sequence (94% identity over 62 basesfrom 423 to 362); embICVOM01010076.1, unannotated whole genome shotgunsequence (100% identity over 62 bp from 62 to 1); ebmICVOM01001690.1,94% identity over 62 bases from 1270 to 1331); gbIGAOU01004675.1,unannotated shotgun transcriptome assembly (99% identity over 697 ntfrom 299 to 995); gbIGAOU01036897.1, unannotated shotgun transcriptomeassembly (100% identity over 358 bp from 358 to 1); gbIGAOU01021755.1,unannotated shotgun transcriptome assembly (100% identity over 304 bpfrom 5 to 308); gbIGAOU01027645.1, unannotated shotgun transcriptomeassembly (100% identity over 102 bp from 831 to 730).

The best nucleotide sequence matches (83% sequence identity with 100%coverage) from species other than red clover are XM_003598989.2 andCU468290.9 from Medicago truncatula. XM_003598989.2 is annotated as“spermidine hydroxycinnamoyl transferase” based on EVidenceModeler geneannotation software. Proteins of these encoded genes are 75% identicalto the proteins (SEQ ID NO: 2 and SEQ ID NO: 4, respectively) encoded bythe cloned red clover HDT cDNAs (SEQ ID NO: 1 and SEQ ID NO: 3,respectively). Other top matches are similarly annotated as spermidinehydroxycinnamoyl transferase, or hydroxycinnamoyl transferase-like. Mostor all annotations do not appear to be based on experimental biochemicaldata, however.

Example 3. Evaluation of HDT mRNA Levels in Red Clover Leaves by ReverseTranscription PCR

Expression of HDT was evaluated using semiquantitative reversetranscribed PCR using cDNA from unexpanded or mature red clover leavesas the template. PCR reactions contained 25 pmol each ms881 (SEQ ID NO:9) and ms882 (SEQ ID NO: 10) (for detection of HDT) or ms171 (SEQ ID NO:17) and ms172 (SEQ ID NO: 18) (for detection of actin, as a control)(see Table 3), cDNA equivalent to 50 ng total RNA, 12.5 μL EconoTaq PlusGreen Mastermix (Lucigen Corp., Middleton, Wis.) and water to make 25μL. Reactions were incubated for 30 sec at 94° C. in the block of athermocycler. This incubation was followed by 25 cycles of 94° C. for 20sec (denaturation), 64° C. for 20 sec (annealing), and 72° C. for 30 sec(extension) followed by a final 2 min extension at 72° C. 3 μL of thePCR reactions were resolved via electrophoresis on a 1.5% agarose gelusing standard techniques.

HDT expression was much higher in unexpanded leaves than in matureleaves. Expression of a control gene, actin, was similar betweenunexpanded and mature leaves. Based on these data, expression of thecloned HDT gene (higher in unexpanded leaves) is consistent with therelative HDT enzyme activity measured in these tissues.

TABLE 3 Desig- nation Sequence (5′ to 3′) ms809TWYTAYCCWDTRGCTGGHMG (SEQ ID NO: 5) ms815AYWGSCYTYCCMYAWCCAAAATC (SEQ ID NO: 6) ms867AMTTCATCAAYWCATGGKC (SEQ ID NO: 7) ms870CCAAAATCWGMWTCRTRAAHVGG (SEQ ID NO: 8) ms881CTGGATACCTAGAACATCTTCTTCATTGGC (SEQ ID NO: 9) ms882CACCTTTGGAGCCACGTTTTGAACACTTGG (SEQ ID NO: 10) ms884CAACACAGAACTTCAASCTAGCATACC (SEQ ID NO: 11) ms885ACCAACTTAGAGGGTGATTTTGGGTC (SEQ ID NO: 12) ms886CGGGCCATGGTAACCATTATAGCTTCTCAC (SEQ ID NO: 13) ms887GGGCCTCGAGTCATATCTCCTCATAAAAATACTTGTT (SEQ ID NO: 14) ms888GTCTAGAAAACAATGGTAACCATTATAGCTTCTCAC (SEQ ID NO: 15) ms889CGGTACCTCATATCTCCTCATAAAAATACTTGTT (SEQ ID NO: 16) ms170GGTGTGAGTCACACTGTGCCAATCT (SEQ ID NO: 17) ms171CGGAACCTCTCAGCTCCAATTGTGA (SEQ ID NO: 18)

Example 4. Enzymatic Activity of HDT1 and HDT2 Produced in E. coli

To determine whether the cloned red clover cDNAs (HDT1 and HDT2) possesshydroxycinnamoyl-CoA:L-DOPA hydroxycinnamoyl transferase activity, theopen reading frames were placed behind the IPTG-inducible promoter ofthe pET28 expression vector and expressed in E. coli. Plasmidscontaining full-length red clover HDT coding regions (both HDT1 (SEQ IDNO: 1) and HDT2 (SEQ ID NO: 3)) were used as templates in PCR reactionswith primers designed to introduce an NdeI restriction site at the startcodons (ms886—SEQ ID NO: 13) and an XhoI site immediately following thestop codon (ms887—SEQ ID NO: 14) of each open reading frame. Theresulting PCR products were digested with NcoI and XhoI and insertedinto pET28a (Novagen, Madison, Wis.) digested with NcoI and XhoI. Theinsert is operably linked to the IPTG-inducible promoter contained inpET28. Each plasmid was sequenced to confirm correct sequence wasgenerated.

Each pET28 derivative containing HDT coding regions (pET28-HDT1 andpET28-HDT2) or pET28 (as a negative control) were transformed,individually, into BL21(DE3)RIL Codon Plus E. coli (AgilentTechnologies, Santa Clara, Calif.). Cultures of E. coli harboringHDT-containing plasmids (BL21/pET28-HDT1; BL21/pET28-HDT2) or controlempty vector plasmid (BL21/pET28) were grown at 37° C. with shaking (225rpm) in TB medium (per L: 12 g tryptone, 24 g yeast extract, 4 mLglycerol, 2.3 g KH₂PO₄, 12.5 K₂HPO₄) supplemented with 50 μg/mLkanamycin and 34 μg/mL chloramphenical to an OD_(600nm) of approximately1.5. Cultures were cooled on ice to approximately 10° C., induced byaddition of isopropyl-beta-D-thiogalactopyranoside (IPTG) to 1 mM, andincubated at 10° C. with shaking (150 rpm) for an additional 20 hours.Cultures were lysed using BugBuster reagent (Novagen) according to themanufacturer's suggested procedures and fractionated into soluble andinsoluble portions. The soluble fraction was isolated, divided intosingle use aliquots, flash frozen in liquid nitrogen and stored at −80°C. until ready to use.

To assess if either HDT1 or HDT2 proteins possessed the anticipatedenzymatic activity, in-vitro reactions for hydroxycinnamoyl-CoAtransferase activity contained 100 mM sodium phosphate buffer (pH 7.5),25 mM ascorbate, 1 mM p-coumaroyl-, caffeoyl-, or feruloyl-CoA donorsubstrate, 1 mM acceptor substrate (L-DOPA or L-tyrosine), and solubleE. coli extract (BL21/pET28-HDT1; BL21/pET28-HDT2; or BL21/pET28).Reactions were incubated at 30° C. for up 1 hour then stopped by theaddition of ⅕ volume of 10% formic acid. Precipitated protein wasremoved by centrifugation (17,000×g for 5 min at room temperature).

The supernatant is analyzed for reaction products by HPLC. Phenolicsamples from the in-vitro reactions were analyzed on a Shim-Pack XR-ODSII (C-18) 120 Å column (Shimadzu Scientific Instruments North America,Columbia, Md., USA; 100×2.0 mm×2.2 micron) using a two solvent system[Solvent A: deionized water with 0.1% (v/v) formic acid, Solvent B:acetonitrile] at a flow rate of 0.5 mL/min. The HPLC conditions were 5min isocratic 2% Solvent B, 10 min gradient to 30% Solvent B, 3 mingradient to 100% Solvent B, 5 min isocratic 100% Solvent B, 0.5 mingradient to 2% Solvent B and 3.5 min isocratic re-equilibration at 2%Solvent B. Compound elution was monitored (250 to 500 nm) with aUV/visible photodiode array detector (PDA). FIG. 4A shows the reversephase HPLC of the in-vitro reaction of E. coli BL21/pET28-HDT1 extractwith caffeoyl-CoA and L-DOPA showing clovamide peak at approximately10.245; FIG. 4B shows the reverse phase HPLC of the in-vitro reaction ofE. coli BL21/pET28 (negative control) with caffeoyl-CoA and L-DOPAshowing no clovamide peak; and FIG. 4C shows reverse phase HPLC of pureclovamide with peak at approximately 10.245. Data for E. coliBL21/pET28-HDT2 is not shown but is similar to the data of BL21/pET-HDT1(FIG. 4A).

In-vitro reactions of extracts of BL21/pET28-HDT1 or BL21/pET28-HDT2with caffeoyl-CoA and L-tyrosine or with p-coumaroyl- or feruloyl-CoAdonors and L-DOPA or L-tyrosine acceptors also produced the expectedproducts. In-vitro reactions with any of the hydroxycinnamoyl-CoA donorsand L-DOPA or L-tyrosine acceptors with BL21/pET28 (negative control)extract failed to produce any hydroxycinnamoyl-amide products.

When expressed in E. coli, the protein products of the cloned HDT cDNAs(both HDT1 and HDT2) are capable of transferring atrans-hydroxycinnamoyl moiety from the corresponding CoA derivative(p-coumaroyl-, caffeoyl-, and feruloyl-CoA) to L-DOPA or L-tyrosineusing similar in-vitro assays as described above. See FIG. 3.

Example 5. Genetically Altered Alfalfa Expressing Red Clover HDT1 orHDT2

For expression of either HDT1 or HDT2 in genetically altered alfalfa,PCR primer pairs were designed to introduce XbaI (ms888—SEQ ID NO: 15)and KpnI (ms889—SEQ ID NO: 16) restriction endonuclease sites flankingthe 5′ and 3′ ends of the coding regions of the two red clover HDT genes(HDT1 and HDT2). Additionally, the forward primer provided the proposeddicot consensus sequence AAACA (Joshi, et al., 1997, Plant Mol. Biol.35:993-1001) immediately upstream of the initiating methionine codon.This primer pair was used in PCR reactions with plasmids containing thefull-length red clover HDT1 or HDT2 coding regions as templates. Theresulting PCR fragments were cloned as XbaI-KpnI fragments downstream ofthe CsVMV promoter (Verdaguer, et al., 1996, Plant Mol. Biol.31:1129-1139) in a derivative of pBIB-HYG plant transformation vector(Becker, 1990, Nucleic Acids Res. 18:203-203; Verdonk and Sullivan,2013, Botany 91:117-122). The cloned inserts were sequenced to confirmthat no mutations occurred.

HDT plant expression constructs containing a selectable marker forhygromycin resistance (pBIB-HYG/HDT1; pBIB-HYG/HDT2) or empty vectorcontaining selectable marker for hygromycin resistance(pBIB-HYG—negative control) were transformed into Agrobacteriumtumefaciens strain LBA4404 (Hellens, et al., 2000, Trends Plant Sci.5:446-451). The resulting A. tumefaciens strains were used togenetically modify a highly regenerable clone of Regen-SY alfalfa(Bingham, 1991) as previously described (Samac and Austin-Phillips,2006, Alfalfa (Medicago sativa L.), in Wang, ed., AgrobacteriumProtocols, 2nd Edition. Humana Press, Totowa, N.J., pp 301-311).Briefly, 12-15 leaves of alfalfa are cut into 6 pieces each. The leafpieces are dipped in a suspension of Agrobacterium containingpBIB-HYG/HDT1, pBIB-HYG/HDT2, or pBIB-HYG. The leaf pieces areco-cultivated with the Agrobacterium for one week on non-selectivemedium. Following co-cultivation, the leaf pieces are transferred tohygromycin containing medium to allow selection of transformed cells.Whole plants are regenerated from transformed cells via somaticembryogenesis using a series of media with differing hormonecompositions. The resulting genetically altered alfalfa have the T-DNAregion of pBIB-HYG/HDT1, pBIB-HYG/HDT2, or pBIB-HYG integrated intotheir genomes. The genetically altered alfalfa were allowed to grow andleaves were harvested.

To assess the accumulation of hydroxycinnamoyl compounds, tissue samplesof each genetically altered alfalfa plant were ground in liquid nitrogenin a mortar and pestle, or for small samples in a 2 mL screw cap tubewith two 4 mm glass beads using a Mini-BeadBeater (Biospec Products,Bartlesville, Okla.). The ground frozen tissue was extracted at roomtemperature with 10 mL/g 100 mM HCl, 50 mM ascorbic acid. Extracts werefiltered through Miracloth (Calbiochem, Billerica, Mass.) or glass woolthen centrifuged at 20,000×g at room temperature. 1 mL of the resultingsupernatant was applied to a 1 mL ENVI-18 solid phase extraction column(Supelco, St. Louis, Mo., USA) pre-equilibrated with 3×1 mL of methanoland 3×1 mL 0.1% acetic acid in water (pH adjusted to 2.5 with HCl). Thecolumn was washed with 3×1 mL 0.1% acetic acid in water (pH adjusted to2.5 with HCl) and eluted with 1 mL methanol.

Tissue was powdered in liquid nitrogen using a mortar and pestle or forsmall samples in a 2 mL screw cap tube with two 4 mm glass beads using aMini-BeadBeater (Biospec Products, Bartlesville, Okla.). The frozenpowdered tissue was added to 1 to 2 mL/g extraction buffer containing100 mM Na phosphate (pH 7.5), 100 mM ascorbic acid (pH adjusted to 7.5with NaOH), and 1% (v/v) protease inhibitor cocktail (P-9599, Sigma, St.Louis, Mo.). The frozen, powdered tissue and buffer were thoroughlymixed by stirring or vortex mixing (depending on amount of tissue andvolume) until the mixture thawed and reached a temperature of 6 to 8° C.The slurry was filtered through a layer of Miracloth (Calbiochem,Billerica, Mass.) on top of a double layer of cheesecloth, as muchliquid as possible was squeezed out, and the filtrate collected on ice.The filtrate was divided among microcentrifuge tubes and centrifuged at17,000×g at 4° C. for 5 min. The supernatant was removed to freshmicrocentrifuge tubes, the centrifugation repeated, and the supernatantretained. Supernatants (typically 30% of the packed column volume) wereapplied to previously prepared spin columns (1-10 mL syringes packedwith Sephadex G-25 Superfine [GE Healthcare, Uppsala, Sweden]equilibrated with 100 mM Na phosphate [pH 7.5 or as specified forindividual experiments], and centrifuged for 1 min at 200×g prior tosample application) to remove low molecular weight compounds, and insome cases, to change the pH of the extract. Following supernatantapplication, the columns were centrifuged for 2 min at 200×g and theflow through (desalted protein extract) retained. Following addition offresh protease inhibitor cocktail (to 0.5% [v/v]), extracts were dividedinto 150 to 200 μL aliquots, flash frozen in liquid nitrogen, and storedat −80° C. until needed. In the case of pH adjustment by the spin columnprocedure, pH was confirmed by spotting a small amount of extract on pHindicator paper. Protein content of the extracts was determined usingBio-Rad Protein Assay (Bio-Rad Laboratories, Hercules, Calif.) usingbovine serum albumin as the standard.

Phenolics from the leaves of the genetically altered alfalfa plantscontaining pBIB-HYG/HDT1, pBIB-HYG/HDT2, or pBIB-HYG were extracted andanalyzed by HPLC with PDA and MS detection. The HPLC protocol used isprovided above. For MS detection, elution was also monitored with aMS2020 mass spectrometer (MS) (Shimadzu Scientific Instruments NorthAmerica) using a dual ion source (electrospray and atmospheric pressurechemical ionization) with data collection in both positive and negativeion modes. MS data was collected between 2.0 and 16.0 min of the HPLCrun, scanning for m/z between 50 and 500 u at 7500 u/sec, with detectorvoltage of 1.3 kV, nebulizing gas flow of 1.5 L/min, drying gas flow of10 L/min, desolvation line and heat block temperatures of 250° C. Whenpeaks were quantified, purchased clovamide or free hydroxycinnamic acidswere used as standards (Nielsen, et al., 1984, Phytochem 23:1741-1743;Sullivan and Zeller, 2012).

Compared to genetically altered plants with only the vector (pBIB-HYG),several of the genetically altered plants containing eitherpBIB-HYG/HDT1 or pBIB-HYG/HDT2 showed the presence of new phenolics. Twoof the detected peaks have m/z=−326 by MS, the expected negative ion ofthe amide that would be formed between p-coumaric acid and L-tyrosine.One of these is indistinguishable from trans-p-coumaroyl-L-tyrosineformed in-vitro using E. coli BL21/pET28-HDT1 in terms of retentiontime, UV absorption spectrum, and m/z. The second m/z=−326 peak islikely the cis isomer, since cis isomers of hydroxycinnamic acidderivatives, especially p-coumaroyl and feruloyl) are known to form inplanta (Sullivan, 2014, Planta 239:1091-1100). Two other of the detectedpeaks have m/z=−356 by MS, the expected negative ion of the amide thatwould be formed between ferulic acid and tyrosine. One of these isindistinguishable from trans-feruloyl-L-tyrosine formed in-vitro usingE. coli BL21/pET28-HDT1 or BL21/pET28-HDT2 in terms of retention time,UV absorption spectrum, and m/z. The second m/z=−356 peak is likely thecis isomer as described above for p-coumaroyl-L-tyrosine. Additionally,when protein extracts were made from of two independent geneticallyaltered alfalfa plants expressing the red clover either HDT1 or HDT2gene, they had enzymatic activity capable of transferring caffeic acidmoieties from caffeoyl-CoA to L-tryosine or L-DOPA. No enzymaticactivity capable of transferring caffeoyl-CoA to L-tyrosine or L-DOPAwas detected in the leaves of a control alfalfa plant transformed withthe empty pBIB-HYG vector. These results indicate that activehydroxycinnamoyl-CoA:L-DOPA/tyrosine hydroxycinnamoyl transferase ispresent in alfalfa when the red clover either HDT1 or HDT2 transgenesare present.

Genetically altered alfalfa containing pBIB-HYG/HDT1 or pBIB-HYG/HDT2are allowed to grow for several months until harvesting. The geneticallyaltered alfalfa are assayed for the present of clovamide and relatedhydroxycinnamoyl amides using LC-MS protocols described above. Thegenetically altered alfalfa contain clovadime, N-caffeoyl-L-tyrosine,N-p-coumaroyl-L-DOPA, and N-feruloyl-L-DOPA. The genetically alteredalfalfa containing pBIB-HYG/HDT1 or pBIB-HYG/HDT2 are also assessed forpost-harvest protein degradation in the presence of PPO (also producedby a transgene) using the protocols set forth in U.S. Pat. No.8,338,339. The genetically altered alfalfa have less post-harvestprotein degradation than occurs in wild-type alfalfa. Thus, clovamideand related hydroxycinnamoyl amides produced by HDT1 and/or HDT2 resultin the genetically altered alfalfa (a forage crop) having a polyphenoloxidase system (PPO) which provides post-harvest protein protection(from degradation).

Example 6. Utilization of HDT1 or HDT2 Sequence for Rapid TraitIntrogression and Accurate Gene Stacking for Clovamide ProductionCoupled with Marker Assisted Selection

Isolation of the HDT1 and HDT2 genes from red clover and the generationof genetically altered alfalfa that contain one of these genes, thegenetically altered alfalfa being able to producehydroxycinnamoyl-CoA:L-DOPA/tyrosine hydroxycinnamoyl transferase andclovamide, has the potential to reduce protein degradation post-harvestin alfalfa. The identification of the cDNA sequences of the HDT1 andHDT2 genes also led to the development of DNA markers which can be usedto screen molecularly altered plants rapidly to improve determine plantscontaining these genes and thus have the phenotype of reduction ofpost-harvest protein degradation. Genetically altered plants can beachieved by rapid introgression of one of the HDT1 and HDT2 genes. Onecan use the primers having SEQ ID NO: 9 or SEQ ID NO: 10 to identifythese genetically altered plants very early in plant development, at twoto three leaf stage, providing great savings in time, space, effort andcost during actual introgression. These primers provide accuracy towardsidentification of genetically altered plants containing either HDT1 orHDT2 gene. Application of correct gene stacks (for example, whencombined with HDT1 or HDT2) and rapid introgression into elite plantlines coupled with marker assay is a valuable application of thediscovery of the HDT1 and HDT2 genes that encodehydroxycinnamoyl-CoA:L-DOPA/tyrosine hydroxycinnamoyl transferase andfor the ability of the genetically altered plant to produce clovamidewhich reduces post-harvest protein degradation.

The foregoing detailed description and certain representativeembodiments and details of the invention have been presented forpurposes of illustration and description of the invention. It is notintended to be exhaustive or to limit the invention to the precise formsdisclosed. It will be apparent to practitioners skilled in the art thatmodifications and variations may be made therein without departing fromthe scope of the invention. All references cited herein are incorporatedby reference.

I, the inventor claim:
 1. A genetically altered alfalfa plant havinghydroxycinnamoyl-CoA:L-DOPA/tyrosine hydroxycinnamoyl transferaseactivity comprising a promoter operably linked to a heterologous cDNAwherein said heterologous cDNA encodes ahydroxycinnamoyl-CoA:L-DOPA/tyrosine hydroxycinnamoyl transferase andwherein said genetically altered alfalfa plant produces saidhydroxycinnamoyl-CoA:L-DOPA/tyrosine hydroxycinnamoyl transferase. 2.The genetically altered alfalfa plant of claim 1 wherein saidhydroxycinnamoyl-CoA:L-DOPA/tyrosine hydroxycinnamoyl transferase has anamino acid sequence selected from the group consisting of SEQ ID NO: 2,SEQ ID NO: 4, a sequence that is at least 95% identical to SEQ ID NO: 2,and a sequence that is at least 95% identical to SEQ ID NO:
 4. 3. Apollen from said genetically altered alfalfa plant of claim
 2. 4. A seedfrom said genetically altered alfalfa plant of claim
 2. 5. A cell fromsaid genetically altered alfalfa plant of claim
 2. 6. The geneticallyaltered alfalfa plant of claim 1 wherein said cDNA has a nucleotidesequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO:3, a sequence that is at least 95% identical to SEQ ID NO: 1, and asequence that is at least 95% identical to SEQ ID NO:
 3. 7. A pollenfrom said genetically altered alfalfa plant of claim
 6. 8. A seed fromsaid genetically altered alfalfa plant of claim
 6. 9. A cell from saidgenetically altered alfalfa plant of claim
 6. 10. A cDNA comprising anucleotide sequence selected from the group consisting of SEQ ID NO: 1,SEQ ID NO: 3, a sequence that is at least 95% identical to SEQ ID NO: 1,and a sequence that is at least 95% identical to SEQ ID NO: 3, whereinthe protein encoded by said cDNA hashydroxycinnamoyl-CoA:L-DOPA/tyrosine hydroxycinnamoyl transferaseactivity.
 11. An expression cassette comprising a promoter operablylinked to a heterologous cDNA, wherein said heterologous cDNA comprisinga nucleotide sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO: 3, a sequence that is at least 95% identical to SEQ ID NO:1, and a sequence that is at least 95% identical to SEQ ID NO: 3,wherein the protein encoded by said cDNA hashydroxycinnamoyl-CoA:L-DOPA/tyrosine hydroxycinnamoyl transferaseactivity.
 12. A cDNA that encodes a hydroxycinnamoyl-CoA:L-DOPA/tyrosinehydroxycinnamoyl transferase comprising a nucleotide sequence thatencodes a hydroxycinnamoyl-CoA:L-DOPA/tyrosine hydroxycinnamoyltransferase wherein said hydroxycinnamoyl-CoA:L-DOPA/tyrosinehydroxycinnamoyl transferase has an amino acid sequence selected fromthe group consisting of SEQ ID NO: 2, SEQ ID NO: 4, a sequence that isat least 95% identical to SEQ ID NO: 2, and a sequence that is at least95% identical to SEQ ID NO:
 4. 13. An expression cassette comprising apromoter operably linked to a heterologous cDNA, wherein saidheterologous cDNA encodes a hydroxycinnamoyl-CoA:L-DOPA/tyrosinehydroxycinnamoyl transferase having an amino acid sequence selected fromthe group consisting of SEQ ID NO: 2, SEQ ID NO: 4, a sequence that isat least 95% identical to SEQ ID NO: 2, and a sequence that is at least95% identical to SEQ ID NO:
 4. 14. A method of reducing post-harvestprotein degradation in a genetically altered alfalfa plant comprising(a) introducing a promoter operably linked to a heterologous cDNA intoan alfalfa plant to provide a genetically altered alfalfa plant, whereinsaid heterologous cDNA encodes a hydroxycinnamoyl-CoA:L-DOPA/tyrosinehydroxycinnamoyl transferase having an amino acid sequence selected fromthe group consisting of SEQ ID NO: 2, SEQ ID NO: 4, a sequence that isat least 95% identical to SEQ ID NO: 2, and a sequence that is at least95% identical to SEQ ID NO: 4, and (b) selecting the genetically alteredalfalfa plant that contains said heterologous cDNA and produces saidhydroxycinnamoyl-CoA:L-DOPA/tyrosine hydroxycinnamoyl transferase,wherein said hydroxycinnamoyl-CoA:L-DOPA/tyrosine hydroxycinnamoyltransferase produces clovamide, at least one related hydroxycinnamoylamide, or a combination thereof, and wherein said clovamide, at leastone related hydroxycinnamoyl amide, or combination thereof reducespost-harvest protein degradation in said genetically altered alfalfa.15. The method of claim 14, wherein said introducing said heterologouscDNA occurs via introgression, breeding, or transfecting an expressioncassette containing said heterologous nucleotide and promoter into saidalfalfa plant.
 16. The method of claim 14 wherein said selecting saidgenetically altered alfalfa plant occurs via marker assisted selection.17. A method of reducing post-harvest protein degradation in agenetically altered alfalfa plant comprising (a) introducing a promoteroperably linked to a heterologous polynucleotide into an alfalfa plantto provide a genetically altered alfalfa plant, wherein saidheterologous polynucleotide comprises the sequence selected from thegroup consisting of SEQ ID NO: 1, SEQ ID NO: 3, a sequence that is atleast 95% identical to SEQ ID NO: 1, and a sequence that is at least 95%identical to SEQ ID NO: 3, and (b) selecting the genetically alteredalfalfa plant that contains said heterologous polynucleotide; whereinsaid heterologous polynucleotide encodes a protein havinghydroxycinnamoyl-CoA:L-DOPA/tyrosine hydroxycinnamoyl transferaseactivity, wherein said genetically altered alfalfa produces said proteinencoded by said heterologous polynucleotide, and wherein said proteinhas hydroxycinnamoyl-CoA:L-DOPA/tyrosine hydroxycinnamoyl transferaseactivity and produces clovamide, at least one related hydroxycinnamoylamide, or a combination thereof, and wherein said clovamide, at leastone related hydroxycinnamoyl amide, or combination thereof reducespost-harvest protein degradation in said genetically altered alfalfa.18. The method of claim 17, wherein said introducing said heterologouspolynucleotide occurs via introgression, breeding, or transfecting anexpression cassette containing said heterologous nucleotide and promoterinto said alfalfa plant.
 19. The method of claim 17 wherein saidselecting said genetically altered alfalfa plant occurs via markerassisted selection.
 20. A method of constructing a genetically alteredalfalfa plant that produces clovamide, at least one relatedhydroxycinnamoyl amide, or a combination thereof comprising (a)introducing a promoter operably linked to a heterologous cDNA into analfalfa plant to provide a genetically altered alfalfa plant, whereinsaid heterologous cDNA encodes a hydroxycinnamoyl-CoA:L-DOPA/tyrosinehydroxycinnamoyl transferase having an amino acid sequence selected fromthe group consisting of SEQ ID NO: 2, SEQ ID NO: 4, a sequence that isat least 95% identical to SEQ ID NO: 2, and a sequence that is at least95% identical to SEQ ID NO: 4, and (b) selecting the genetically alteredalfalfa plant that contains said heterologous cDNA and produces saidhydroxycinnamoyl-CoA:L-DOPA/tyrosine hydroxycinnamoyl transferase,wherein said hydroxycinnamoyl-CoA:L-DOPA/tyrosine hydroxycinnamoyltransferase produces clovamide, at least one related hydroxycinnamoylamide, or a combination thereof.
 21. The method of claim 20, whereinsaid introducing said heterologous cDNA occurs via introgression,breeding, or transfecting an expression cassette containing saidheterologous nucleotide and promoter into said alfalfa plant.
 22. Themethod of claim 20 wherein said selecting said genetically alteredalfalfa plant occurs via marker assisted selection.
 23. A method ofconstructing a genetically altered alfalfa plant that producesclovamide, at least one related hydroxycinnamoyl amide, or a combinationthereof comprising (a) introducing a promoter operably linked to aheterologous polynucleotide into an alfalfa plant to provide agenetically altered alfalfa plant, wherein said heterologouspolynucleotide comprises the sequence selected from the group consistingof SEQ ID NO: 1, SEQ ID NO: 3, a sequence that is at least 95% identicalto SEQ ID NO: 1, and a sequence that is at least 95% identical to SEQ IDNO: 3, and (b) selecting the genetically altered alfalfa plant thatcontains said heterologous polynucleotide; wherein said heterologouspolynucleotide encodes a protein havinghydroxycinnamoyl-CoA:L-DOPA/tyrosine hydroxycinnamoyl transferaseactivity; wherein said genetically altered alfalfa produces said proteinhaving hydroxycinnamoyl-CoA:L-DOPA/tyrosine hydroxycinnamoyl transferaseactivity; and wherein said protein havinghydroxycinnamoyl-CoA:L-DOPA/tyrosine hydroxycinnamoyl transferaseactivity produces said clovamide, at least one related hydroxycinnamoylamide, or a combination thereof.
 24. The method of claim 23, whereinsaid introducing said heterologous polynucleotide occurs viaintrogression, breeding, or transfecting an expression cassettecontaining said heterologous nucleotide and promoter into said alfalfaplant.
 25. The method of claim 23 wherein said selecting saidgenetically altered alfalfa plant occurs via marker assisted selection.26. A kit for determining if an alfalfa plant contains HDT1 or HDT2 geneand thereby produces a hydroxycinnamoyl-CoA:L-DOPA/tyrosinehydroxycinnamoyl transferase, said kit comprising at least one pair ofpolynucleotides; an identifying dye; and instructions for using said atleast one pair of polynucleotides to determine said alfalfa plantcontains said HDT1 or HDT2 gene; and wherein said at least one pair ofsaid polynucleotides have the sequence of SEQ ID NO: 9 and SEQ ID NO:10.